The present invention relates to the field of biotechnology, and more specifically to the field of genomic modification. Disclosed herein are compositions, vectors, and methods of use thereof, for the generation of transgenic cells, tissues, plants, and animals. The compositions, vectors, and methods of the present invention are also useful in gene therapy techniques.
Permanent genomic modification has been a long sought after goal since the discovery that many human disorders are the result of genetic mutations that could, in theory, be corrected by providing the patient with a non-mutated gene. Permanent alterations of the genomes of cells and tissues would also be valuable for research applications, commercial products, protein production, and medical applications. Furthermore, genomic modification in the form of transgenic animals and plants has become an important approach for the analysis of gene function, the development of disease models, and the design of economically important animals and crops.
A major problem with many genomic modification methods associated with gene therapy is their lack of permanence. Life-long expression of the introduced gene is required for correction of genetic diseases. Indeed, sustained gene expression is required in most applications, yet current methods often rely on vectors that provide only a limited duration of gene expression. For example, gene expression is often curtailed by shut-off of integrated retroviruses, destruction of adenovirus-infected cells by the immune system, and degradation of introduced plasmid DNA (Anderson, W F, Nature 329:25-30, 1998; Kay, et al, Proc. Natl. Acad. Sci. USA 94:12744-12746, 1997; Verma and Somia, Nature 389:239-242, 1997). Even in shorter-term applications, such as therapy designed to kill tumor cells or discourage regrowth of endothelial tissue after restenosis surgery, the short lifetime of gene expression of current methods often limits the usefulness of the technique.
One method for creating permanent genomic modification is to employ a strategy whereby the introduced DNA becomes part of (i.e., integrated into) the existing chromosomes. Of existing methods, only retroviruses provide for efficient integration. Retroviral integration is random, however, thus the added gene sequences can integrate in the middle of another gene, or into a region in which the added gene sequence is inactive. In addition, a different insertion is created in each target cell. This situation creates safety concerns and produces an undesirable loss of control over the procedure.
Adeno-associated virus (AAV) often integrates at a specific region in the human genome. However, vectors derived from AAV do not integrate site-specifically due to deletion of the toxic rep gene (Flotte and Carter, Gene Therapy 2:357-362, 1995; Muzyczk, Curr. Topics Microbiol. Immunol. 158:97-129, 1992). The small percentage of the AAV vector population that eventually integrates does so randomly. Other methods for genomic modification include transfection of DNA using calcium phosphate co-precipitation, electroporation, lipofection, microinjection, protoplast fusion, particle bombardment, or the Ti plasmid (for plants). All of these methods produce random integration at low frequency. Homologous recombination produces site-specific integration, but the frequency of such integration is very low.
Another method that has been considered for the integration of heterologous nucleic acid fragments into a chromosome is the use of a site-specific recombinase (an example using Cre is described below). Site-specific recombinases catalyze the insertion or excision of nucleic acid fragments. These enzymes recognize relatively short, unique nucleic acid sequences that serve for both recognition and recombination. Examples include Cre (Sternberg and Hamilton, J Mol Biol 150:467-486, 1981), Flp (Broach, et al, cell-29:227-234, 1982) and R (Matsuzaki, et al, J Bacteriology 172:610-618, 1990).
One of the most widely studied site-specific recombinases is the enzyme Cre from the bacteriophage P1. Cre recombines DNA at a 34 basepair sequence called loxP, which consists of two thirteen basepair palindromic sequences flanking an eight basepair core sequence. Cre can direct site-specific integration of a loxP-containing targeting vector to a chromosomally placed loxP target in both yeast and mammalian cells (Sauer and Henderson, New Biol 2:441-449, 1990). Use of this strategy for genomic modification, however, requires that a chromosome first be modified to contain a loxP site (because this sequence is not known to occur naturally in any organism but P1 bacteriophage), a procedure which suffers from low frequency and unpredictability as discussed above. Furthermore, the net integration frequency is low due to the competing excision reaction also mediated by Cre. Similar concerns arise in the conventional use of other, well-known, site-specific recombinases.
A need still exists, therefore, for a convenient means by which chromosomes can be permanently modified in a site-specific manner. The present invention addresses that need.
Accordingly, in one embodiment, the present invention is directed to a method of site-specifically integrating a polynucleotide sequence of interest in a genome of a eucaryotic cell. The method comprises introducing (i) a circular targeting construct, comprising a first recombination site and the polynucleotide sequence of interest, and (ii) a site-specific recombinase into the eucaryotic cell, wherein the genome of the cell comprises a second recombination site native to the genome and recombination between the first and second recombination sites is facilitated by the site-specific recombinase. The cell is maintained under conditions that allow recombination between the first and second recombination sites and the recombination is mediated by the site-specific recombinase. The result of the recombination is site-specific integration of the polynucleotide sequence of interest in the genome of the eucaryotic cell.
The recombinase may be introduced into the cell before, concurrently with, or after introducing the circular targeting construct. Further, the circular targeting construct may comprise other useful components, such as a bacterial origin of replication and/or a selectable marker.
In certain embodiments, the recombinase may facilitate recombination between two sites designated recombinase-mediated-recombination sites (RMRS) and the RMRS comprises a first DNA sequence (RMRS5xe2x80x2), a core region A, and a second DNA sequence (RMRS3xe2x80x2) in the relative order RMRS5xe2x80x2-core region A-RMRS3xe2x80x2. In this embodiment, for example, RMRS may be a loxP site or a FRT site and the recombinase may be Cre and FLP, respectively.
In additional embodiments,(i) the second recombination site is a pseudo-RMRS site, and the second recombination site comprises a first DNA sequence (attT5xe2x80x2), a core region B, and a second DNA sequence (attT3xe2x80x2) in the relative order attT5xe2x80x2-core region B-attT3xe2x80x2, and (ii) the first recombination site is a hybrid-recombination site comprising RMRS5xe2x80x2-core region B-RMRS3xe2x80x2 or attT5xe2x80x2-core region B-attT3xe2x80x2.
In yet further embodiments, the site-specific recombinase is a recombinase encoded by a phage selected from the group consisting of xcfx86C31, TP901-1, and R4. The recombinase may facilitate recombination between a bacterial genomic recombination site (attB) and a phage genomic recombination site (attP), and (i) the second recombination site may comprise a pseudo-attP site, and (ii) the first recombination site may comprise the attB site or (i) the second recombination site may comprise a pseudo-attB site, and (ii) the first recombination site may comprise the attP site.
In another embodiment, (i) attB comprises a first DNA sequence (attB5xe2x80x2), a bacterial core region, and a second DNA sequence (attB3xe2x80x2) in the relative order attB5xe2x80x2-bacterial core region-attB3xe2x80x2, (ii) attP comprises a first DNA sequence (attP5xe2x80x2), a phage core region, and a second DNA sequence (attP3xe2x80x2) in the relative order attP5xe2x80x2-phage core region-attP3xe2x80x2, and (iii) wherein the recombinase meditates production of recombination-product sites that can no longer act as a substrate for the recombinase, the recombination-product sites comprising the relative order attB5xe2x80x2-recombination-product site-attP3xe2x80x2 and attP5xe2x80x2-recombination-product site-attB3xe2x80x2.
In particularly preferred embodiments, (i) the second recombination site is a pseudo-attP site, the second recombination site comprises a first DNA sequence (attT5xe2x80x2), a core region B, and a second DNA sequence (attT3xe2x80x2) in the relative order attT5xe2x80x2-core region B-attT3xe2x80x2, (ii) the first recombination site is an attB site comprising attB5xe2x80x2-bacterial core region-attB3xe2x80x2, and (iii) wherein the recombinase meditates production of recombination-product sites that can no longer act as a substrate for the recombinase, the recombination-product sites comprising the relative order attT5xe2x80x2-recombination-product site-attB3xe2x80x2{polynucleotide of interest}attB5xe2x80x2-recombination-product site-attT3xe2x80x2. Alternatively, (i) the second recombination site is a pseudo-attB site, and the second recombination site comprises a first DNA sequence (attT5xe2x80x2), a core region B, and a second DNA sequence (attT3xe2x80x2) in the relative order attT5xe2x80x2-core region B-attT3xe2x80x2, (ii) the first recombination site is an attP site comprising attP5xe2x80x2-bacterial core region-attP3xe2x80x2, and (iii) wherein the recombinase meditates production of recombination-product sites that can no longer act as a substrate for the recombinase, the recombination-product sites comprising the relative order attT5xe2x80x2-recombination-product site-attP3xe2x80x2{polynucleotide of interest}attP5xe2x80x2-recombination-product site-attT3xe2x80x2.
In yet further embodiments, the site-specific recombinase is introduced into the cell as a polypeptide. In alternative embodiments, the site-specific recombinase in introduced into the cell as a polynucleotide encoding the recombinase and an expression cassette, optionally carried on a transient expression vector, comprises the polynucleotide encoding the recombinase.
In another embodiment, the invention is directed to a vector for site-specific integration of a polynucleotide sequence into the genome of a eucaryotic cell. The vector comprises (i) a circular backbone vector, (ii) a polynucleotide of interest operably linked to a eucaryotic promoter, and (iii) a first recombination site, wherein the genome of the cell comprises a second recombination site native to the genome and recombination between the first and second recombination sites is facilitated by a site-specific recombinase.
In certain embodiments, the recombinase normally facilitates recombination between a bacterial genomic recombination site (attB) and a phage genomic recombination site (attP) and the first recombination site may be either attB or attP.
In still another embodiment, the invention is directed to a kit for site-specific integration of a polynucleotide sequence into the genome of a eucaryotic cell. The kit comprises, (i) a vector as described above and (ii) a site-specific recombinase.
In another embodiment, the invention is directed to a eucaryotic cell having a modified genome. The modified genome comprises an integrated polynucleotide sequence of interest whose integration was mediated by a recombinase and wherein the integration was into a recombination site native to the eucaryotic cell genome and the integration created a recombination-product site comprising the polynucleotide sequence.
In certain embodiments, the recombination-site product comprises the components attT5xe2x80x2-recombination-product site-attB3xe2x80x2 and attB5xe2x80x2-recombination-product site-attT3xe2x80x2, wherein (i) the native recombination site is a pseudo-attP site, and the native recombination site comprises a first DNA sequence (attT5xe2x80x2), a core region B, and a second DNA sequence (attT3xe2x80x2) in the relative order attT5xe2x80x2-core region B-attT3xe2x80x2, (ii) the integrated polynucleotide sequence comprises a first recombination site comprising an attB site comprising attB5xe2x80x2-bacterial core region-attB3xe2x80x2, and (iii) wherein the recombinase meditates production of recombination-product sites that can no longer act as a substrate for the recombinase, the recombination-product sites comprising the relative order attT5xe2x80x2-recombination-product site-attB3xe2x80x2{polynucleotide of interest}attB5xe2x80x2-recombination-product site-attT3xe2x80x2. Alternatively, the recombination-site product comprises the components attT5xe2x80x2-recombination-product site-attB3xe2x80x2 and attB5xe2x80x2-recombination-product site-attT3xe2x80x2, wherein (i) the native recombination site is a pseudo-attB site, and the native recombination site comprises a first DNA sequence (attT5xe2x80x2), a core region B, and a second DNA sequence (attT3xe2x80x2) in the relative order attT5xe2x80x2-core region B-attT3xe2x80x2, (ii) the integrated polynucleotide sequence comprises a first recombination site comprising an attP site comprising attP5xe2x80x2-phage core region-attP3xe2x80x2, and (iii) wherein the recombinase meditates production of recombination-product sites that can no longer act as a substrate for the recombinase, the recombination-product sites comprising the relative order attT5xe2x80x2-recombination-product site-attP3xe2x80x2{polynucleotide of interest}attP5xe2x80x2-recombination-product site-attT3xe2x80x2.
In further embodiments, the subject invention is directed to transgenic plants and animals comprising at least one cell as described above, as well as methods of producing the same.
In yet other embodiments, the invention is directed to methods of treating a disorder in a subject in need of such treatment. The method comprises site-specifically integrating a polynucleotide sequence of interest in a genome of at least one cell of the subject, wherein the polynucleotide facilitates production of a product that treats the disorder in the subject. The site-specific integration may be carried out in vivo in the subject, or ex vivo in cells and the cells are then introduced into the subject.
A further embodiment of the invention comprises cells, tissues, transgenic animals and/or plants whose genomes have been modified using the methods described herein.
In another aspect, the present invention provides a method of modifying a genome of a cell. In the method, an attB or an attP recombination site is into the genome of a cell, wherein (i) the recombination site is recognized by a recombinase, and (ii) the cell normally does not comprise the attB or attP site. The vectors described herein and above are useful in the practice of this aspect of the invention. In a preferred embodiment, the cell that is being modified is a eucaryotic cell.
In yet another aspect, the present invention provides expression cassettes, comprising a polynucleotide encoding a site-specific recombinase, wherein (i) the recombinase is encoded by a phage (typically selected from the group consisting of xcfx86C31, TP901-1, and R4) and the recombinase is operably linked to a eucaryotic promoter. The vectors described herein and above are useful in the practice of this aspect of the invention.
These and other embodiments of the present invention will readily occur to those of ordinary skill in the art in view of the disclosure herein.